Cerebrovascular reactivity to carbon dioxide tension in newborns: data from combined time-resolved near-infrared spectroscopy and diffuse correlation spectroscopy

Abstract. Significance Critically ill newborns are at risk of brain damage from cerebrovascular disturbances. A cerebral hemodynamic monitoring system would have the potential role to guide targeted intervention. Aim To obtain, in a population of newborn infants, simultaneous near-infrared spectroscopy (NIRS)-based estimates of cerebral tissue oxygen saturation (StO2) and blood flow during variations of carbon dioxide tension (pCO2) levels within physiologic values up to moderate permissive hypercapnia, and to examine if the derived estimate of metabolic rate of oxygen would stay constant, during the same variations. Approach We enrolled clinically stable mechanically ventilated newborns at postnatal age >24  h without brain abnormalities at ultrasound. StO2 and blood flow index were measured using a non-invasive device (BabyLux), which combine time-resolved NIRS and diffuse-correlation spectroscopy. The effect of changes in transcutaneous pCO2 on StO2, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen index (tCMRO2i) were estimated. Results Ten babies were enrolled and three were excluded. Median GA at enrollment was 39 weeks and median weight 2720 g. StO2 increased 0.58% (95% CI 0.55; 0.61, p<0.001), CBF 2% (1.9; 2.3, p<0.001), and tCMRO2 0.3% (0.05; 0.46, p=0.017) per mmHg increase in pCO2. Conclusions BabyLux device detected pCO2-induced changes in cerebral StO2 and CBF, as expected. The small statistically significant positive relationship between pCO2 and tCMRO2i variation is not considered clinically relevant and we are inclined to consider it as an artifact.


Introduction
Cerebrovascular disturbances are involved in the pathogenesis of brain damage in critically ill newborns.The risk is increased if autoregulation, i.e., the ability of the cerebral vasculature to compensate for changes in perfusion pressure, is impaired. 1However, reactivity of cerebral blood flow (CBF) to fluctuations in carbon dioxide tension is a normal feature of the cerebral vasculature; it is fully operational in newborn and preterm infants 2 and is likely to play a key role, potentially exposing the vulnerable developing brain to ischemic insults when hypocapnia occurs. 3Conversely, hypercapnia increases CBF and intracranial pressure; the vasodilator action of carbon dioxide is quick and more potent than that of any chemical agent.
A continuous monitor of cerebral hemodynamics and oxygen metabolism would have the potential to guide individualized care and targeted intervention to reduce the risk of cerebral hypoxia-ischemia.
Continuous wave (CW) spatially resolved near-infrared spectroscopy (SR-NIRS) allows measurement, albeit with a relatively poor precision, of cerebral tissue oxygen saturation (StO 2 ), which has been proposed as a surrogate of CBF.However, this approach relies on the assumption of a stable oxygen consumption, which is dependent on the local tissue demand as well as on perfusion and oxygen carrying capacity (delivery).Time-resolved reflectance spectroscopy (TRS) overcomes some assumptions that are necessary to measure regional tissue oxygenation by SR-NIRS, and diffusion correlation spectroscopy (DCS) can be used to assess microvascular CBF (calculated as blood flow index-BFI). 4he European-funded BabyLux (BBLX) project (EU CIP ICT PSP n. 620996) aimed to develop a non-invasive and cot-sided device that combines TRS, measuring regional oxygenation with improved precision, and DCS, assessing regional tissue perfusion. 5,6By combining these two measures, estimation of regional oxygen metabolism could be also provided.
We aimed at using the BabyLux device 4 to obtain simultaneous hybrid NIRS-based estimates of cerebral StO 2 and CBF when carbon dioxide tension levels are adjusted by manipulation of mechanical ventilation in clinically stable newborn infants without brain pathology.The purpose is to examine if the derived estimate of cerebral metabolic rate of oxygen would stay constant, as predicted, when the changes in carbon dioxide tension are small and within physiological limits up to moderate hypercapnia.

Study Population and Protocol
The trial (registered at ClinicalTrials.gov -NCT02815618) was conducted at Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy and it was approved by the local research ethics committee and by the Italian Medical Device Agency.
Patients were enrolled from 2017 to 2018 and the analysis of data and preparation of the draft were delayed for lack of protected research time for the clinical investigators during COVID pandemic.
Inclusion criteria were: clinically stable newborns of any gestational age at birth (defined by stable pulse oximeter saturation -SpO 2in the normal range for gestational age -GA) undergoing invasive mechanical ventilation with normal cranial ultrasound and postnatal age >24 h.Signed informed parental consent was obtained before enrollment.
Monitoring of cerebral hemodynamics and oxygenation: the BabyLux sensor (Fig. 1) was placed on one side of fronto-parietal region, held in place by a black self-adhesive elastic bandage; monitoring with INVOS technology was performed, according to clinical practice, with adhesive sensor (Neonatal Oxyalert TM NIRSensor 5100 INVOS technology) placed on the other side of fronto-parietal region.SpO 2 and carbon dioxide tension (pCO 2 ) estimated by transcutaneous measurement (tcpCO 2 ) were continuously monitored in all infants (Radiometer ® ).
As an arterial or capillary gas sample was drawn on clinical indication, tcpCO 2 monitoring was adjusted to pCO 2 and changes in ventilatory settings were introduced according to routine clinical practice to normalize pCO 2 in case of either (slight-to-moderate) hypercapnia or hypocapnia.Spontaneous changes in tcpCO 2 were also recorded.Changes in tcpCO 2 of at least 4 mmHg occurring within the following minutes were analyzed.
For each infant the following parameters were recorded: SpO 2 , StO 2 (measured by both BabyLux -StO 2 -BBLX-and INVOS Medtronic -StO 2 -INVOS-), and BFI measured by BabyLux.Tissue oxygen extraction (TOE) and cerebral metabolic rate of oxygen index (CMRO 2 i) were calculated from the BabyLux signals.Considering that CMRO 2 is calculated using the TOE and that TOE, which is calculated from StO 2 , is higher than the cerebro-venous saturation due to the arterial component in the StO 2 signal, we named it tissue cerebral metabolic rate of oxygen index-tCMRO 2 i.BFI was then converted into CBF with the conversion factor estimated by Giovannella et al. 7 that is rCBF ¼ 0.89ðml∕100 g∕minÞ∕ðcm 2 ∕sÞ × 10 9 × BFI, with (0.56, 1.39) ðml∕100 g∕minÞ∕ðcm 2 ∕sÞ as the corresponding limits of agreement for the conversion factor; tCMRO 2 was then calculated (see Table 1).

Instrumentation
The BabyLux device integrates TRS and DCS modules, both using NIR light.In brief, TRS has potentiality to separate the absorption and scattering coefficients allowing for absolute Fig. 1 The BabyLux device.measurements, and to utilize time-gating of path-lengths to emphasize signals from deeper tissues, whereas DCS relies on the interaction between long coherence laser light and moving scatters allowing a measurement of red blood cell movement module.The TRS module employs pulsed lasers operating at three different wavelengths centred at about 685, 760, and 820 nm, respectively.The DCS module uses a CW long coherence laser at 785 nm with an output power <20 mW.TRS and DCS share a compact and light-weight 5iberoptic probe, for injection and collection of the light signals into the tissue with source-detector separation of 15 mm.Sampling frequency was 1 Hz."For the calculation of BFI, 10 s moving average of μ a at 760 nm was used to reduce noise propagation from TRS to DCS analysis and a fixed sample average estimate of μ s 0 was used (μ s 0 ¼ 7 cm −1 ); BFI and μ s 0 are indeed coupled in the equation describing the intensity field autocorrelation curve, 8 and an eventual error in the latter is propagated in an error in the BFI. 9 Therefore, using an individual estimation of μ s 0 can increase the interindividual variability for the BFI." (For details on instrumentation, data processing, and quality assessment, see previously published papers. 4,10,11).

Data Analysis
BFI and StO 2 reactivity to changes in tcpCO 2 were calculated over periods of clinical stability in terms of steady SpO 2 .
The effect of changes in tcpCO 2 on the measured variables (StO 2 -BBLX and StO 2 -INVOS, BFI, CBF, TOE, tCMRO 2 i, and tCMRO 2 ) was estimated using linear mixed-effect models, which are appropriate in settings in which repeated measurements are made on the same statistical unit (infant/subject), with the subject considered as random effect.We averaged all measurements to the unit of the minute, and we used all the monitored data series per patient to study the optical∕tcpCO 2 relationship.Summary statistics for each variable are then presented.Both induced and spontaneous tcpCO 2 changes were included in the same models.Model results are expressed as regression coefficients (changes per mmHg change of tcpCO 2 ), 95% CI and p-values.
BBLX and INVOS oximeter values were compared using the Bland-Altman plot for repeated measures, 12 allowing for within-subject correlation, and a mixed effect model with infant considered as random effect was used to estimate the mean oxygenation level-dependent bias.We also presented the Spearman's rank correlation coefficient between the absolute values of the two measurements.Values of p < 0.05 were considered statistically significant.Logarithmic transformation (natural log) of BFI and tCMRO 2 i raw data was done to normalize the right skewed distribution of residuals.
Statistical analyses were performed using R, version 3.4.3(R Foundation for Statistical Computing, Vienna, Austria).

Results
Ten babies were enrolled from February 2017 to March 2018.Three infants were excluded from the analysis (due to the following reasons: changes in tcpCO 2 < 4 mmHg in one case (Table 3, infant 10), bad positioning of the probe resulting in high variability in optical parameters with estimated values of the scattering coefficient < 4 cm −1 in the another one (Table 3, infant 3), and SpO 2 instability, together with high variability of optical parameters in the third one (Table 3, infant 2), The mean analyzed time for each infant was 21.04 min (sd 8.16).
Table 2 summarizes the clinical and biochemical characteristics of the study population while Table 3 shows μ a (cm −1 ) and μ 0 s (cm −1 ) values of the ten enrolled infants.Table 4 illustrates the measured and calculated variables.Mixed models' results showed that StO 2 -INVOS, StO 2 -BBLX, BFI, CBF, tCMRO 2 i, and tCMRO 2 all had a positive relationship with tcpCO 2 ; on the contrary, TOE was negatively related to tcpCO 2 .Table 5 shows changes per mmHg variation in tcpCO 2 for each variable.The coefficient derived from the model for log(CBF) is 0.021, which corresponds to a change of ½expð0.021Þ− 1% ¼ 2.12% in CBF per mmHg and the coefficient derived from the model for logðtCMRO 2 Þ is 0.003, which corresponds to a change of ½expð0.003Þ− 1% ¼ 0.30% in tCMRO 2 per mmHg.The estimates have narrow confidence limits.
In Fig. 2, we present the changes of the studied variable (StO 2 -BBLX, TOE, CBF, and tCMRO 2 ) according to tcpCO 2 variation in each of the seven infants.The absolute value of StO 2 -INVOS on the average was 12.4% higher than the value of StO 2 -BabyLux, and the mean difference (MD) in the individual infants ranged from 0.1% to 19.5%; within infants, the BabyLux and INVOS oximeter values were highly correlated, Spearmans' correlation coefficients ranged from 0.68 to 0.91 (Fig. 3).
The average oxygenation level-dependent bias between the devices was small and statistically insignificant at 0.021% per % (p ¼ 0.113, mixed effect model), but the 95% confidence interval for bias in the individual infants was quite wide, −1.15% to 1.2% per % (Fig. 2).

Discussion
This study demonstrates that bedside and non-invasive TRS combined with DCS detects pCO 2induced changes in cerebral StO 2 and CBF as expected.Numbers indicated in bold correspond to the excluded infants due to high variability in optical parameters with estimated values of the scattering coefficient <4 cm −1 .
The confidence intervals of the estimates of effect are narrow, but the small number of subjects is a limitation and made us abstain from analysis of interactions with other factors that may affect cerebrovascular response, such as analgesics, perfusion pressure, and brain maturity.This is the first study in which DCS, which has been previously qualitatively validated and in-vivo calibrated, 6 was used in clinically stable mechanically ventilated newborn infants to measure cerebrovascular reactivity to tcpCO 2 variation within a range of normal values.
Indeed, cerebrovascular reactivity (CBF and CMRO 2 ) were already measured by hybrid DCS and NIRS during hypercapnia in unstable neonates with congenital heart disease and validated by concurrent magnetic resonance imaging (MRI) data. 13,14he BabyLux device allows measurement of BFI, expressed in cm 2 ∕s, which has been demonstrated to be proportional to blood flow in the tissue. 4,8We measured lower BFI values compared with previously published studies in which DCS was used to measure microvascular CBF in healthy term newborns (mean; range: 9.85 × 10 −9 ; 2.20 × 10 −9 , 30.00 × 10 −9 -versus range 15 to 45 × 10 −9 cm 2 ∕ sec). 15Higher BFI values were evident even in cases in which data were obtained with the same BabyLux method from 9.85 × 10 −9 compared to 27 × 10 −9 cm 2 ∕ sec in 23 healthy full terms). 16However, this was not the case when BFI was converted into CBF.
In fact, to compare estimation of CBF and oxygen metabolism obtained by other methods, we converted BFI measurements (cm 2 ∕s) into flow units (ml∕100 g∕ min) using a previously validated conversion formula derived from a neonatal piglet model in which DCS was validated    against 15 O-water Positron Emission Tomography (see Ref. 7 for further details), which showed a good agreement btween calculated and expected values. 7he calculated CBF values in the present study are consistent with the ones reported in a population of 12 mechanically ventilated term infants (10 AE 4.7 versus 11.9 AE 4.9 ml∕100 g∕ min) measured by 133 Xe clearance (Pryds et al. 17 ) and the ones reported in healthy term (10 AE 4.7 versus 13.4 AE 4.2 ml∕100 g∕min) by Liu et al. 18 and preterm infants at term by De Vis et al. (10 AE 4.7 versus 14 AE 3 ml∕100 g∕min) using MRI techniques. 19BF showed the expected positive relationship with tcpCO 2 , although at only 2% per mmHg increase in pCO 2 .Thus, the reactivity to CO 2 was less than previously estimated by DCS in neonates with congenital heart defects (end-tidal CO 2 ) 14 and by 133 Xe clearance in mechanically ventilated preterm babies <33 weeks GA (tcpCO 2 ). 2 The novelty of the study relies on the simultaneous measurement of cerebral StO 2 and CBF, which allowed calculation of cerebral oxygen metabolism (see Table 1

for conversion).
The calculated values of tCMRO 2 (0.52 mlO 2 ∕100 g∕min) were lower compared to previous studies: neonates in intensive care units at different gestational ages 20,21 (∼1.0 ml O 2 ∕ 100 g∕min) and healthy and non-sedated neonates aged between 35 and 42 gestational weeks (0.76 mlO 2 ∕100 g∕min, 18 0.60 mlO 2 ∕100 g∕min, 19 and 1.2 mlO 2 ∕100 g∕min 22 ).This difference can partly be explained by the fact that StO 2 overestimates cerebro-venous saturation due to the arterial contribution to the NIRS signal.If 33% of the NIRS signal comes from arterial blood and 66% from venous blood (assuming that the capillary blood volume is negligeable, thus corresponding to an a-v-ratio of 1:2), the "real CMRO 2 would be þ50% (0.78), and if 25% of the signal comes from arterial blood (a-v ratio of 1:3), the real CMRO 2 " would be þ33% (0.69).Furthermore, CMRO 2 in mechanically ventilated newborn infants may be reduced, as CBF has been shown to be. 3 This is the first study that analyzed variation of tCMRO 2 in stable mechanically ventilated infants as induced by changes in pCO 2 .In this study, pCO 2 was kept in a permissive range (up to modearate hypercapnia) and we therefore expected to find a constant tCMRO 2 . 8However, we found a statistically significant increase with increasing pCO 2 .In a preclinical study on macaque monkeys exposed to CO 2 inhalation, Zappe et al. observed a reduction of neuronal activity with increasing pCO 2 . 23This would rather suggest a reduction in CMRO 2 based on the strong correlation between CMRO 2 and brain's electrical activity. 246][27] CO 2 -induced decrease of brain pH is likely to result in changes in membrane permeability of cortical cells and reduced excitatory postsynaptic activity. 26Furthermore, an effect on the un-loading of oxygen from haemoglobin at the capillary level (as a result of the Bohr effect on the oxygen-haemoglobin dissociation curve) may limit the increase in StO 2 at high levels of pCO 2 .All of this would also suggest a reduction rather than an increase in CMRO 2 at high pCO 2 .
Therefore, we are inclined to believe that the positive association of pCO 2 and CMRO 2 in our study is an artifact.The possibilities for artefacts are an overestimation of the reactivity of StO 2 or an underestimation of the reactivity of CBF, or an increase in the a-v ratio-or a combination.Actually, an increase in the a-v ratio has been demonstrated during hypoxia 28 and during hypovolemic hypotension. 29Since CO 2 is a vasodilator on the arterial side, and if the venous side is not passively dilated as much, then this would be a simple explanation for our findings.
The statistically significant positive relationship between tcpCO 2 and tCMRO 2 variation, however, was very small (1 ‰ change per mmHg tcpCO 2 ) and may hardly be considered clinically relevant when compared with the 20% reduction in CMRO 2 reported in term infants suffering hypoxic-ischemic encephalopathy (0.48 mlO 2 ∕100 g∕ min versus 0.60 mlO 2 ∕100 g∕ min) in healthy term infants. 19egarding the comparison between INVOS and BabyLux, it is well known that the INVOS neonatal sensor gives higher values than other devices 30 and our data confirm this observation.The inter-individual variability was very high; however, our measurements were obtained by single placement for each of the two sensors and it has been demonstrated that placementreplacement reproducibility for cerebral oximeters is relatively poor. 31The large inter-individual variability in oxygenation-level dependent bias among the infants was surprising.However, it has been previously demonstrated, in preterm infants simultaneously monitored with INVOS and NONIN devices, both with neonatal sensors, that the response to drops in SpO 2 due to apnea

Fig. 3
Fig. 3 Bland-Altman plots of StO 2 -BabyLux and StO 2 -INVOS values for each infant.The MD of the simultaneous values (y -axis) over their mean (x -axis) has been plotted.The black line in the middle of the graph represents the MD between the two methods.The dashed lines indicate 95% CI limits of agreement.In infant 1, 2, and 5, the bias is clearly dependent on the level of oxygenation, decreasing or increasing.

Fig. 2
Fig. 2 Relationship between tcpCO 2 and the studied variables (StO 2 -BBLX, TOE, CBF, and tCMRO 2 ).The CBF-CO 2 reactivity corresponds to a change of 2.1% per mmHg increase in pCO 2 , whereas the CMRO 2 -CO 2 reactivity corresponds to a change of 0.3% per mmHg increase in pCO 2 .Each colour indicates a single infant: dots represent the values of the studied variable for each value of tcpCO 2 ; lines represent the linear regression model for each infant.Log = natural log (light blue line infant 1, purple line infant 4, orange line infant 5, red line infant 6, green line infant 7, blue line infant 8, and gray line infant 9).

Table 1
Measured and calculated variables.

Table 2
Characteristics of the study population.

Table 5
Mixed effect models estimate for each variable per mmHg variation in tcpCO 2 .

Table 4
Descriptive statistics for the measured and calculated variables.Mean and standard deviation (SD) are computed from subjects' means (N ¼ 7) calculated on the entire period.tcpCO 2 , transcutaneous carbon dioxide tension; SpO 2 , steady pulse oximeter saturation; StO 2 , tissue oxygen saturation; TOE, tissue oxygen extracion; BFI, blood flow index; CBF, cerebral blood flow; and tCMRO 2 i, tissue cerebral metabolic rate